DNA Methylation of Human Choline Kinase Alpha Promoter-Associated CpG Islands in MCF-7 Cells

Choline kinase (CK) is the enzyme catalyzing the first reaction in CDP-choline pathway for the biosynthesis of phosphatidylcholine. Higher expression of the α isozyme of CK has been implicated in carcinogenesis, and inhibition or downregulation of CKα (CHKA) is a promising anticancer approach. This study aimed to investigate the regulation of CKα expression by DNA methylation of the CpG islands found on the promoter of this gene in MCF-7 cells. Four CpG islands have been predicted in the 2000 bp promoter region of ckα (chka) gene. Six CpG island deletion mutants were constructed using PCR site-directed mutagenesis method and cloned into pGL4.10 vectors for promoter activity assays. Deletion of CpG4C region located between –225 and –56 significantly increased the promoter activity by 4-fold, indicating the presence of important repressive transcription factor binding site. The promoter activity of methylated full-length promoter was significantly lower than the methylated CpG4C deletion mutant by 16-fold. The results show that DNA methylation of CpG4C promotes the binding of the transcription factor that suppresses the promoter activity. Electrophoretic mobility shift assay analysis showed that cytosine methylation at MZF1 binding site in CpG4C increased the binding of putative MZF1 in nuclear extract. In conclusion, the results suggest that DNA methylation decreased the promoter activity by promoting the binding of putative MZF1 transcription factor at CpG4C region of the ckα gene promoter.

Download Full-text

Isolation of Estrogen-Responsive Genes with a CpG Island Library

Molecular and Cellular Biology ◽

10.1128/mcb.18.1.442 ◽

1998 ◽

Vol 18 (1) ◽

pp. 442-449 ◽

Cited By ~ 87

Author(s):

Toru Watanabe ◽

Satoshi Inoue ◽

Hisahiko Hiroi ◽

Akira Orimo ◽

Hiroyuki Kawashima ◽

...

Keyword(s):

Estrogen Receptor ◽

Binding Site ◽

Target Genes ◽

Cpg Island ◽

Cpg Islands ◽

Human Cells ◽

Receptor Protein ◽

Upstream Region ◽

Oxidase Subunit ◽

Mcf 7

ABSTRACT In order to isolate novel estrogen-responsive genes, we utilized a CpG island library in which the regulatory regions of genes are enriched. CpG islands were screened for the ability to bind to a recombinant estrogen receptor protein with a genomic binding site (GBS) cloning method. Six CpG islands were selected, and they contained perfect, imperfect, and/or multiple half-palindromic estrogen-responsive elements (EREs). Northern blot analysis of various human cells showed that all these genomic fragments hybridized to specific mRNAs, suggesting that the genes associated with these EREs might be transcribed in human cells. Then cDNAs associated with two of them, EB1 and EB9, were isolated from libraries of human placenta and MCF-7 cells derived from a human breast cancer, respectively. Both transcripts were increased by estrogen in MCF-7 cells. The increase is inhibited by actinomycin D but not by cycloheximide, indicating that no protein synthesis is required for the up-regulation. The cDNA associated with EB1 encodes a 114-amino-acid protein similar to the cytochrome c oxidase subunit VIIa, named COX7RP (cytochromec oxidase subunit VII-related protein). The cDNA associated with EB9 is homologous only to an express sequence tag and was named EBAG9 (estrogen receptor-binding fragment-associated gene 9). The palindromic ERE of EB1 is located in an intron of COX7RP, and that of EB9 is in the 5′ upstream region of the cDNA. Both EREs had significant estrogen-dependent enhancer activities in a chloramphenicol acetyltransferase assay, when they were inserted into the 5′ upstream region of the chicken β-globin promoter. We therefore propose that the CpG-GBS method described here for isolation of the DNA binding site from the CpG island library would be useful for identification of novel target genes of certain transcription factors.

Download Full-text

Aberrant silencing of the CpG island-containing human O6-methylguanine DNA methyltransferase gene is associated with the loss of nucleosome-like positioning.

Molecular and Cellular Biology ◽

10.1128/mcb.17.10.5813 ◽

1997 ◽

Vol 17 (10) ◽

pp. 5813-5822 ◽

Cited By ~ 36

Author(s):

S A Patel ◽

D M Graunke ◽

R O Pieper

Keyword(s):

Transcription Factor ◽

Chromatin Structure ◽

Dna Methyltransferase ◽

Cytosine Methylation ◽

Cpg Island ◽

Cpg Islands ◽

Transcription Factor Binding ◽

Open Chromatin ◽

Factor Binding ◽

Methylguanine Dna Methyltransferase

Tumor-associated aberrant silencing of CpG island-containing genes has been correlated with increased cytosine methylation, a "closed" chromatin structure, and exclusion of transcription factor binding in the CpG island/promoter regions of affected genes. Given the lack of understanding of what constitutes a closed chromatin structure in CpG islands, however, it has been difficult to assess the relationship among cytosine methylation, chromatin structure, and inappropriate gene silencing. In this study, nuclease accessibility analysis was used to more clearly define the chromatin structure in the CpG island of the human O6-methylguanine DNA methyltransferase (MGMT) gene. Chromatin structure was then related to in vivo DNA-protein interactions and cytosine methylation status of the MGMT CpG island in human glioma cells varying in MGMT expression. The results of these studies indicated that the "open" chromatin structure associated with the MGMT CpG island in MGMT+ cells consisted of an approximately 250-bp transcription factor-binding, nuclease-accessible, nucleosome-free region of DNA, whose formation was associated with at least four flanking, precisely positioned nucleosome-like structures. In MGMT- cells, this precise nucleosomal array was lost and was replaced by randomly positioned nucleosomes (i.e., the closed chromatin structure), regardless of whether methylation of the CpG island was spread over the entire island or limited to regions outside the transcription factor binding region. These results suggest that CpG islands facilitate the expression of housekeeping genes by facilitating nucleosomal positioning and that the conditions that alter the formation of this array (such as perhaps methylation) may indirectly affect CpG island-containing gene expression.

Download Full-text

Silencing of the Gene for the α-Subunit of Human Chorionic Gonadotropin by the Embryonic Transcription Factor Oct-3/4

Molecular Endocrinology ◽

10.1210/mend.11.11.9971 ◽

1997 ◽

Vol 11 (11) ◽

pp. 1651-1658 ◽

Cited By ~ 1

Author(s):

Limin Liu ◽

Douglas Leaman ◽

Michel Villalta ◽

R. Michael Roberts

Keyword(s):

Transcription Factor ◽

Binding Site ◽

Gene Promoter ◽

Site Directed Mutagenesis ◽

Mobility Shift ◽

Α Subunit ◽

Choriocarcinoma Cells ◽

Human Choriocarcinoma Cells ◽

Electrophoretic Mobility Shift Assays ◽

Jar Cells

Abstract CG is required for maintenance of the corpus luteum during pregnancy in higher primates. As CG is a heterodimeric molecule, some form of coordinated control must be maintained over the transcription of its two subunit genes. We recently found that expression of human CG β-subunit (hCGβ) in JAr human choriocarcinoma cells was almost completely silenced by the embryonic transcription factor Oct-3/4, which bound to a unique ACAATAATCA octameric sequence in the hCGβ gene promoter. Here we report that Oct-3/4 is also a potent inhibitor of hCG α-subunit (hCGα) expression in JAr cells. Oct-3/4 reduced human GH reporter expression from the −170 hCGα promoter in either the presence or absence of cAMP by about 70% in transient cotransfection assays, but had no effect on expression from either the −148 hCGα or the −99 hCGα promoter. Unexpectedly, no Oct-3/4-binding site was identified within the −170 to −148 region of the hCGα promoter, although one was found around position −115 by both methylation interference footprinting and electrophoretic mobility shift assays. Site-directed mutagenesis of this binding site destroyed the affinity of the promoter for Oct-3/4, but did not affect repression of the promoter. Therefore, inhibition of hCGα gene transcription by Oct-3/4 appears not to involve direct binding of this factor to the site responsible for silencing. When stably transfected into JAr cells, Oct-3/4 reduced the amounts of both endogenous hCGα mRNA and protein by 70–80%. Oct-3/4 is therefore capable of silencing both hCGα and hCGβ gene expression. We suggest that as the trophoblast begins to form, reduction of Oct-3/4 expression permits the coordinated onset of transcription from the hCGα and hCGβ genes.

Download Full-text

Synergy between the NF-E1 erythroid-specific transcription factor and the CACCC factor in the erythroid-specific promoter of the human porphobilinogen deaminase gene

Molecular and Cellular Biology ◽

10.1128/mcb.10.7.3838-3842.1990 ◽

1990 ◽

Vol 10 (7) ◽

pp. 3838-3842

Author(s):

J Frampton ◽

M Walker ◽

M Plumb ◽

P R Harrison

Keyword(s):

Transcription Factor ◽

Base Pair ◽

Promoter Activity ◽

Transient Transfection ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Porphobilinogen Deaminase ◽

Specific Transcription Factor ◽

Specific Manner ◽

Two Factors

A 114-base-pair promoter fragment of the human porphobilinogen deaminase gene functioned in an erythroid-specific manner in transient transfection experiments. Site-directed mutagenesis of the binding site for the erythroid-specific transcription factor (NF-E1) or an adjacent CACCC motif abolished the promoter activity. Increasing the spacing between these sites progressively reduced promoter activity, but there was no evidence that a critical alignment of the two factors on the DNA helix was required.

Download Full-text

Regulatory effect of heat shock transcription factor-1 gene on heat shock proteins and its transcriptional regulation analysis in small abalone Haliotis diversicolor

BMC Molecular and Cell Biology ◽

10.1186/s12860-020-00323-9 ◽

2020 ◽

Vol 21 (1) ◽

Author(s):

Xin Zhang ◽

Yuting Li ◽

Yulong Sun ◽

Mingxing Guo ◽

Jianjun Feng ◽

...

Keyword(s):

Transcription Factor ◽

Heat Shock ◽

Binding Site ◽

Promoter Activity ◽

Luciferase Activity ◽

Haliotis Diversicolor ◽

Shock Response ◽

Flanking Region ◽

Small Abalone ◽

Shock Proteins

Abstract Background The effects of diverse stresses ultimately alter the structures and functions of proteins. As molecular chaperones, heat shock proteins (HSPs) are a group of highly conserved proteins that help in the refolding of misfolded proteins and the elimination of irreversibly damaged proteins. They are mediated by a family of transcription factors called heat shock factors (HSFs). The small abalone Haliotis diversicolor is a species naturally distributed along the southern coast of China. In this study, the expression of HdHSF1 was inhibited by RNAi in hemocytes in order to further elucidate the regulatory roles of HdHSF1 on heat shock responsive genes in abalone. Meanwhile, to understand the transcriptional regulation of the HdHSF1 gene, the 5′-upstream regulatory region of HdHSF1 was characterized, and the relative promoter activity was examined by dual-luciferase reporter gene assay system in HEK293T cell lines. Results After the inhibition of the H. diversicolor HSF1 gene (HdHSF1) by dsRNA (double-stranded RNA), the expression of most heat shock related-genes was down-regulated (p < 0.05). It indicated the importance of HdHSF1 in the heat shock response of H. diversicolor. Meanwhile, 5′-flanking region sequence (2633 bp) of the HdHSF1 gene was cloned; it contained a putative core promoter region, TATA box, CAAT box, CpG island, and many transcription elements. In HEK293T cells, the 5′-flanking region sequence can drive expression of the enhanced green fluorescent protein (EGFP), proving its promoter function. Exposure of cells to the high-temperature (39 °C and 42 °C) resulted in the activation of HdHSF1 promoter activity, which may explain why the expression of the HdHSF1 gene participates in heat shock response. Luciferase activity of different recombinant plasmids, which contained different truncated promoter fragments of the HdHSF1 gene in HEK293T cells, revealed the possible active regions of the promoter. To further identify the binding site of the critical transcription factor in the region, an expression vector with the site-directed mutation was constructed. After being mutated on the GATA-1 binding site, we found that the luciferase activity was significantly increased, which suggested that the GATA-1 binding site has a certain weakening effect on the activity of the HdHSF1 promoter. Conclusions These findings suggest that GATA-1 may be one of the transcription factors of HdHSF1, and a possible signaling pathway mediated by HdHSF1 may exist in H. diversicolor to counteract the adverse effects of heat shock stress.

Download Full-text

DNA methylation of antisense noncoding RNA in the INK locus (ANRIL) is associated with coronary artery disease in a Chinese population

Scientific Reports ◽

10.1038/s41598-019-51921-3 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

Chen-Hui Zhao ◽

Hai-Tao Cao ◽

Jing Zhang ◽

Qiao-Wei Jia ◽

Feng-Hui An ◽

...

Keyword(s):

Coronary Artery Disease ◽

Dna Methylation ◽

Transcription Factor ◽

Coronary Artery ◽

Kegg Pathway ◽

Cpg Island ◽

Density Lipoprotein ◽

Lipoprotein Cholesterol ◽

Pathway Enrichment Analysis ◽

Artery Disease

Abstract To explore the association between methylation of antisense non-coding RNA in the INK4 locus (ANRIL) and coronary artery disease (CAD) development. Methylation levels of ANRIL in 100 subjects with CAD and 100 controls were quantitatively analyzed using Sequenom MassARRAY. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was used to identify novel pathways. Our analyses indicated that 7 to 8 CpG sites within the 2nd CpG island located upstream of ANRIL, also known as cyclin-dependent kinase inhibitor 2B – antisense 1 (CDKN2B-AS1), are hyper-methylated in CAD subjects compared to controls (p = 0.034). The 40th CpG site within the 2nd CpG island located upstream of CDKN2B-AS1 was methylated to a lesser extent in CAD subjects compared to controls (p = 0.045). Both Pearson and Spearman analyses indicated that methylation levels were significantly associated with total cholesterol (r = 0.204, p = 0.004), fasting high-density lipoprotein cholesterol (r = 0.165, p = 0.020), and fasting low-density lipoprotein cholesterol (r = 0.265, p = 0.000). KEGG pathway analysis revealed a significant enrichment of genes associated with the tumor necrosis factor (TNF) signaling pathway. Among them, CCAAT/enhancer binding protein (C/EBPβ) was identified as a key transcription factor that promotes expression of CDKN2B-AS1 through promotor interaction. DNA methylation of the ANRIL promoter was significantly associated with CAD development in our study. Our analyses suggest that C/EBPβ is a key transcription factor that promotes CDKN2B-AS1 expression by directly interacting with the gene promotor mediated by TNF signaling.

Download Full-text

Characterization of Global DNA Methylation in Different Gene Regions Reveals Candidate Biomarkers in Pigs with High and Low Levels of Boar Taint

Veterinary Sciences ◽

10.3390/vetsci7020077 ◽

2020 ◽

Vol 7 (2) ◽

pp. 77 ◽

Cited By ~ 1

Author(s):

Xiao Wang ◽

Haja N. Kadarmideen

Keyword(s):

Dna Methylation ◽

Cpg Island ◽

Cpg Islands ◽

Gene Interaction ◽

Boar Taint ◽

Reduced Representation ◽

Gene Interaction Networks ◽

Reduced Representation Bisulfite Sequencing ◽

Pathways Analysis ◽

Porcine Gene

DNA methylation of different gene components, including different exons and introns, or different lengths of exons and introns is associated with differences in gene expression. To investigate the methylation of porcine gene components associated with the boar taint (BT) trait, this study used reduced representation bisulfite sequencing (RRBS) data from nine porcine testis samples in three BT groups (low, medium and high BT). The results showed that the methylation levels of the first exons and first introns were lower than those of the other exons and introns. The first exons/introns of CpG island regions had even lower levels of methylation. A total of 123 differentially methylated promoters (DMPs), 194 differentially methylated exons (DMEs) and 402 differentially methylated introns (DMIs) were identified, of which 80 DMPs (DMP-CpGis), 112 DMEs (DME-CpGis) and 166 DMIs (DMI-CpGis) were discovered in CpG islands. Importantly, GPX1 contained one each of DMP, DME, DMI, DMP-CpGi, DME-CpGi and DMI-CpGi. Gene-GO term relationships and pathways analysis showed DMP-CpGi-related genes are mainly involved in methylation-related biological functions. In addition, gene–gene interaction networks consisted of nodes that were hypo-methylated GPX1, hypo-methylated APP, hypo-methylated ATOX1, hyper-methylated ADRB2, hyper-methylated RPS6KA1 and hyper-methylated PNMT. They could be used as candidate biomarkers for reducing boar taint in pigs, after further validation in large cohorts.

Download Full-text

DNA Methylation of Endoglin Pathway Genes in Pregnant Women With and Without Preeclampsia

Epigenetics Insights ◽

10.1177/2516865720959682 ◽

2020 ◽

Vol 13 ◽

pp. 251686572095968

Author(s):

Allison H Rietze ◽

Yvette P Conley ◽

Dianxu Ren ◽

Cindy M Anderson ◽

James M Roberts ◽

...

Keyword(s):

Dna Methylation ◽

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

Cpg Island ◽

Methylation Status ◽

Cpg Islands ◽

Control Participant ◽

Sample Collection ◽

Promoter Regions ◽

Participant Group

Objective: We compared blood-based DNA methylation levels of endoglin ( ENG) and transforming growth factor beta receptor 2 ( TGFβR2) gene promoter regions between women with clinically-overt preeclampsia and women with uncomplicated, normotensive pregnancies. Methods: We used EpiTect Methyl II PCR Assays to evaluate DNA methylation of CpG islands located in promoter regions of ENG (CpG Island 114642) and TGFβR2 (CpG Island 110111). Preeclampsia was diagnosed based on blood pressure, protein, and uric acid criteria. N = 21 nulliparous preeclampsia case participants were 1:1 frequency matched to N = 21 nulliparous normotensive control participants on gestational age at sample collection (±2 weeks), smoking status, and labor status at sample collection. Methylation values were compared between case and control participant groups [( ENG subset: n = 20 (9 cases, 11 controls); TGFβR2 subset: n = 28 (15 cases, 13 controls)]. Results: The majority of the preeclampsia cases delivered at ⩾34 weeks’ gestation (83%). Average methylation levels for ENG ([M ± (SD)]; Case Participant Group = 6.54% ± 4.57 versus Control Participant group = 4.81% ± 5.08; P = .102) and TGFβR2 (Case Participant Group = 1.50% ± 1.37 vs Control Participant Group = 1.70% ± 1.40; P = .695) promoter CpG islands did not differ significantly between the participant groups. Removal of 2 extreme outliers in the ENG analytic subset revealed a trend between levels of ENG methylation and pregnancy outcome (Case Participant Group = 5.17% ± 2.16 vs Control Participant Group = 3.36% ± 1.73; P = .062). Conclusion: Additional epigenetic studies that include larger sample sizes, investigate preeclampsia subtypes, and capture methylation status of CpG island shores and shelves are needed to further inform us of the potential role that ENG and TGFβR2 DNA methylation plays in preeclampsia pathophysiology.

Download Full-text

Transcription Factor ZBED6 Mediates IGF2 Gene Expression by Regulating Promoter Activity and DNA Methylation in Myoblasts

Scientific Reports ◽

10.1038/srep04570 ◽

2014 ◽

Vol 4 (1) ◽

Cited By ~ 11

Author(s):

Yong-Zhen Huang ◽

Liang-Zhi Zhang ◽

Xin-Sheng Lai ◽

Ming-Xun Li ◽

Yu-Jia Sun ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Transcription Factor ◽

Promoter Activity ◽

Igf2 Gene

Download Full-text

Transcription Regulation by Tandem-Bound FNR at Escherichia coli Promoters

Journal of Bacteriology ◽

10.1128/jb.185.20.5993-6004.2003 ◽

2003 ◽

Vol 185 (20) ◽

pp. 5993-6004 ◽

Cited By ~ 17

Author(s):

Anne M. L. Barnard ◽

Jeffrey Green ◽

Stephen J. W. Busby

Keyword(s):

Escherichia Coli ◽

Transcription Factor ◽

Binding Site ◽

Transcription Regulation ◽

Transcription Start Site ◽

Promoter Activity ◽

Start Site ◽

Transcription Start ◽

Substitution Mutant

ABSTRACT FNR is an Escherichia coli transcription factor that regulates the transcription of many genes in response to anaerobiosis. We have constructed a series of artificial FNR-dependent promoters, based on the melR promoter, in which a consensus FNR binding site was centered at position −41.5 relative to the transcription start site. A second consensus FNR binding site was introduced at different upstream locations, and promoter activity was assayed in vivo. FNR can activate transcription from these promoters when the upstream FNR binding site is located at many different positions. However, sharp repression is observed when the upstream-bound FNR is located near positions −85 or −95. This repression is relieved by the FNR G74C substitution mutant, previously identified as being defective in transcription repression at the yfiD promoter. A parallel series of artificial FNR-dependent promoters, carrying a consensus FNR binding site at position −61.5 and a second upstream DNA site for FNR, was also constructed. Again, promoter activity was repressed by FNR when the upstream-bound FNR was located at particular positions.

Download Full-text